Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.


Introduction
Human cytomegalovirus (HCMV) is a common virus which infects 40 to 80% of individuals in the human population [1]. The virus is usually acquired during early life when the innate and adaptive immune systems are not fully mature [2], with the eye being one of the major target organs. Thus, the incidence of HCMV chorioretinitis is reported to be 25% in infants with symptomatic congenital HCMV infection [3][4][5][6]. Furthermore, the choroid/RPE may be a site of HCMV latency, since recent studies in our laboratory of ocular tissue from human cadavers have revealed that HCMV DNA was present in 17% (4 of 24) of choroid/RPE samples [7].
In order to investigate the effects of lifelong infection with cytomegalovirus, we established an in vivo mouse model in which systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spreads to the eye, with subsequent establishment of

SD-OCT (Spectral-Domain Optical Coherence Tomography) Analysis
Six eyes from the MCMV latently infected mice at 18 months p.i. and three eyes from age-matched uninfected controls were used for sequencing analysis. Prior to removal of the eyes, SD-OCT examinations were performed, and retinal thickness was calculated using a Leica Envisu R2210 system (Bioptigen, Leica, Morrisville, NC, USA). Compared to agematched uninfected control eyes, a significantly reduced retinal thickness was observed in all six eyes of the infected mice ( Figure 1B) as previously reported [8], while severe photoreceptor (PRC) degeneration, including disappearance of the entire outer nuclear layer (ONL) in some areas, was observed in three of six eyes ( Figure 1A). In addition, one choroidal neovascularization (CNV)-like lesion was observed in one of these three eyes with severe PRC degeneration (not shown).
The number of genes (≥1 normalized count) expressed in each sample ranged from 12,875 to 14,230 with a cutoff of |fold change| ≥ 2 and q ≤ 0.05 used to identify differentially expressed genes (DEGs). Compared to the uninfected control eyes (samples c1, c2, c3), 321 DEGs (208 downregulated and 113 upregulated, Supplemental Table S1) were identified in the six virus latently infected eyes. The top 15 upregulated and downregulated genes are listed in Table 1, together with the fold changes and FDR values. Among virus-infected eyes, 48 DEGs (38 downregulated and 10 upregulated, Supplemental Table  S2) were identified in three eyes with severe retinal degeneration (samples h1, h2, h3), (C) Validation of RNA-seq transcriptome data by qRT-PCR analysis of eight genes in three control and six virus-infected samples. (D) A Heat map for three control samples (c1, c2, c3) and six virus-infected samples (m1, m2, m3, h1, h2, h3).
The number of genes (≥1 normalized count) expressed in each sample ranged from 12,875 to 14,230 with a cutoff of |fold change| ≥ 2 and q ≤ 0.05 used to identify differentially expressed genes (DEGs). Compared to the uninfected control eyes (samples c1, c2, c3), 321 DEGs (208 downregulated and 113 upregulated, Supplementary Table S1) were identified in the six virus latently infected eyes. The top 15 upregulated and downregulated genes are listed in Table 1, together with the fold changes and FDR values. Among virusinfected eyes, 48 DEGs (38 downregulated and 10 upregulated, Supplementary Table S2) were identified in three eyes with severe retinal degeneration (samples h1, h2, h3), com-

Validation of Differentially Expressed Genes
RNA-seq transcriptome data were validated by qRT-PCR analysis and expression levels of eight genes were analyzed in three control and six virus-infected samples. As shown in Figure 1C, similar expression trends were observed by both qRT-PCR analysis and RNA-Seq for all eight genes. A correlation (R2) of 0.8904 was observed between the log2FC of ∆CT values derived by qRT-PCR analysis and the log 2 FC value derived by RNA-seq analysis.

Analysis of Canonical Pathways
QIAGEN IPA analysis was used to identify involvement of canonical pathways with a p-value of less than 10 −2 . These pathways, together with the differentially expressed genes in each canonical pathway, are listed in Table 2. Among a total of 17 canonical pathways identified, 10 function in neuroretinal signaling, with the majority of differentiated regulated genes involved in these pathways being downregulated in infected eyes. As shown in Figure 2, for example, all 22 differentially expressed genes linked to the Phototransduction Pathway of rod and cone cells, were downregulated. These include genes involved in cAMP and PKA-mediated signaling, which participate in the modulation of presynaptic GABA release [38] and are themselves regulated by Relaxin signaling in a biphasic manner [39]. cAMP signaling also plays a role in regulating glutamatergic transmission via phosphorylation of certain ionotropic glutamate receptors [40] through dopamine-DARPP32 feedback. Since DARPP-32 is localized in horizontal cells, amacrine cells, and Müller glial cells, this may result in the disfunction of these important retinal cell types. Expression of genes involved in RNA splicing was also downregulated in infected eyes, consistent with previous results which have shown that RNA processing defects are associated with many diseases of the neuron, including retinal degeneration [41]. Other pathways affected include the Endothelin-1 (ET-1) pathway, which was also downregulated, and G beta gamma signaling. The neuropeptide ET-1 is expressed in multiple retinal cell types, including RPE, photoreceptors, the inner plexiform layer, and ganglion cell layer [42] and functions in neuromodulation and neurotransmission [42,43]. However, ET-1 could also activate ET receptors in the retinal and choroidal vasculature, suggesting an important role in regulating in situ blood flow [42]. G beta gamma signaling plays a critical role in rod cell function in low light conditions [44], as well as neuronal CREB signaling, which is one of the major regulators of neurotrophin responses [45,46], and G-protein coupled receptor signaling.
In contrast, the transcription of genes involved in several immune response pathways was upregulated. These included pathways involved in NFAT-regulated dendritic cell maturation, phagosome formation, and communication between innate and adaptive immune cells. Upregulated gene transcription was also observed in the GP6 signaling pathway, which functions in platelet activation and thrombus formation [47]. The Tec kinase signaling pathway, which regulates lymphocyte development, activation, and differentiation [48], as well as in the opioid signaling pathway, which is critically involved in many physiological processes including neuroprotection and immune response [49][50][51].

Analysis of Diseases and Functions
The QIAGEN IPA analysis also permitted the categorization of differentially expressed genes according to known disease associations and function as listed below.

1.
Ophthalmic Disease, Organismal Injury, and Visual System Function. Many differentially expressed genes were noted in sections of this category. As shown in Table 3, differential expression of genes implicated in retinal degeneration, including degeneration of the photoreceptor, rod, and outer segments of the cone cell, was detected with z scores greater than 2. Seven differentially expressed genes were noted with a z score of 1.568. In addition, differential expression of genes required for maintenance of retinal cell and photoreceptor function and quantity were noted with z scores less than −2. Table 3. Ophthalmic disease and visual system function.

Diseases or Functions Annotation p-Value
Activation z-Score  Table 4, many differentially expressed genes implicated in the death of retinal cells and epithelial tissues, either by apoptosis or necrosis, were detected. TUNEL assays and Western blots were used to confirm this observation. As shown in Figure 3A, many TUNEL-positive cells were observed in the choroid (indicated by white arrows), RPE layer (indicated by arrow heads), and outer nuclear layer (indicated by red arrows) in virus-infected eyes, while in contrast, only a few TUNEL-positive cells were observed in the outer nuclear layer of eyes from age-matched uninfected mice. Eyes of MCMV neonatally infected mice, as well as the eyes of the age-matched, uninfected controls, were also analyzed by Western blotting. As shown in Figure 3B, MCMV infection was associated with increased production of cleaved caspase 3, RIP3, MLKL, and decreased production of rhodopsin, indicating that both apoptosis and necroptosis may contribute to cell death. outer nuclear layer (indicated by red arrows) in virus-infected eyes, while in contrast, only a few TUNEL-positive cells were observed in the outer nuclear layer of eyes from age-matched uninfected mice. Eyes of MCMV neonatally infected mice, as well as the eyes of the age-matched, uninfected controls, were also analyzed by Western blotting. As shown in Figure 3B, MCMV infection was associated with increased production of cleaved caspase 3, RIP3, MLKL, and decreased production of rhodopsin, indicating that both apoptosis and necroptosis may contribute to cell death.   3. Cellular Movement and Immune Cell Trafficking. As shown in Table 5, significant changes in the transcription of genes involved in migration, infiltration, and activation of multiple immune cell types were observed in infected eyes. Immunostaining was used to confirm these observations. As shown in Figure 4, accumulation of Iba1 positive macrophage/microglia and GFAP positive Müller cells/glia was observed in the subretinal space and outer nuclear layer of the eyes of aged, infected mice, but not in age-matched uninfected controls. tion of multiple immune cell types were observed in infected eyes. Immunostaining was used to confirm these observations. As shown in Figure 4, accumulation of Iba1 positive macrophage/microglia and GFAP positive Müller cells/glia was observed in the subretinal space and outer nuclear layer of the eyes of aged, infected mice, but not in age-matched uninfected controls.

Analysis of Upstream Regulators
Pathway analysis identified 36 upstream regulators of these differentially expressed genes with activation Z scores of greater than 1.5 and p-values of overlap less than 0.05 ( Table 6). The majority of these upstream regulators are cytokines (IL1, IFN, IL17A, TNF,

Analysis of Upstream Regulators
Pathway analysis identified 36 upstream regulators of these differentially expressed genes with activation Z scores of greater than 1.5 and p-values of overlap less than 0.05 ( Table 6). The majority of these upstream regulators are cytokines (IL1, IFN, IL17A, TNF, etc.) or transcription factors (STAT1, STAT3) that are involved in innate immunity/inflammation. Several growth factors (VEGF, TGF) were also identified as activated upstream regulators. As shown in the summary graph ( Figure 5), upstream regulators, such as IL1, OSM, IL17A, and STAT1, stimulate activation and migration of immune cells, tissue degeneration, and antigen presentation.

Comparison of Infected Eyes with and without Severe Retinal Degeneration
Compared to infected eyes without severe retinal degeneration, we identified 48 DEGs in three infected eyes with severe retinal degeneration (Supplemental Table S2). As shown in Table 7, the majority of differentially regulated genes were involved in pathways related to neuroretinal signaling, cell death, and retinal degeneration. In contrast, no significant differences were detected in immune response pathways, such as those involved in migration, infiltration, or activation of immune cells, between infected eyes with and without severe retinal degeneration.

Discussion
The data presented here complements our earlier studies on lifelong MCMV latency in the retina. These studies demonstrated that systemic MCMV infection of newborn mice resulted in ocular pathology later in life which exhibited some similarities to AMD [8].

Comparison of Infected Eyes with and without Severe Retinal Degeneration
Compared to infected eyes without severe retinal degeneration, we identified 48 DEGs in three infected eyes with severe retinal degeneration (Supplementary Table S2). As shown in Table 7, the majority of differentially regulated genes were involved in pathways related to neuroretinal signaling, cell death, and retinal degeneration. In contrast, no significant differences were detected in immune response pathways, such as those involved in migration, infiltration, or activation of immune cells, between infected eyes with and without severe retinal degeneration.

Discussion
The data presented here complements our earlier studies on lifelong MCMV latency in the retina. These studies demonstrated that systemic MCMV infection of newborn mice resulted in ocular pathology later in life which exhibited some similarities to AMD [8]. Although no infectious virus could be isolated from the retina during latent ocular infection, many gross pathological changes in the retinal architecture were observed. The transcriptional data presented here provides a theoretical framework for understanding retinal and choroidal pathologies in latently infected eyes and demonstrates widespread alterations in the expression of genes critical for maintaining retinal form and function. Thus, systemic virus infection of neonates results in disruption of normal ocular gene expression patterns even in advanced age.
The studies presented herein support the idea that cytomegalovirus ocular latency could be associated with in situ inflammation. Transcription of many inflammatory molecules was elevated, and pathway analysis indicated that several inflammatory pathways, including IL1, STAT1, IL17A, and OSM, were activated with activation Z scores greater than 2 and p-values of overlap less than 0.05. These inflammatory molecules may activate immune cells including macrophages/microglia and could also induce degeneration of photoreceptor, RPE, or choriocapillaris, either directly or indirectly, via activation of immune cells. Our results also demonstrate that multiple cell death pathways, including caspase-3 dependent apoptosis, necrosis, and necroptosis are activated and contribute to degeneration of photoreceptors, RPE, and choroidal capillaries, although future studies are still needed to determine which pathways contribute to which aspects of retinal degeneration.
Our previous studies have shown that during systemic neonatal MCMV infection of 129S1/SvImJ (129S1) mice [7], MCMV spreads to the eye with subsequent establishment of latency in the choroid/RPE. Unlike the neonatal infection of BALB/c mice, only a few MCMV genes are expressed, and no remarkable retinal or choroidal pathology, such as deposits, degeneration of the choriocapillaris, RPE, or photoreceptors, was observed [7]. BALB/c mice, which are susceptible to light damage due to a lack of melanin [52,53], exhibited photoreceptor degeneration (infiltrating cells, loss of outer segments, and decreased retinal thickness) in aged, uninfected control mice in both our own previous studies [8], as well as those of Bell and colleagues [54]. Thus, melanin in the RPE may prevent light-induced retinal damage by absorbing most of the light passing through the pupil and helping to scavenge free radicals [53,55,56], thereby protecting photoreceptors from oxidative stress [57,58]. Melanin might also prevent toxicity through its anti-oxidative function [59] and could play a role in protecting choroidal blood vessels from light damage [60]. Therefore, the susceptibility of BALB/c mice to light damage [61][62][63][64] may be due to an accumulation of reactive oxygen species (ROS) and subsequent oxidative stress and inflammation [65,66]. Previous studies have suggested that oxidative stress mediates the initial activation of viral gene expression during cytomegalovirus latency [67,68]. Following light damage, oxidative stress in MCMV latently infected BALB/c mice could activate expression of ocular virus genes, which in turn might promote production of inflammatory/angiogenic factors, thereby facilitating development of retinal and choroidal pathologies.
The choroid/RPE may be a site of HCMV latency since we have shown that HCMV DNA is present in some human choroid/RPE samples [7]. However, whether HCMV ocular latency contributes to human AMD remains to be determined. AMD is a complex multifactorial disease, and the majority of risk factors, including genetics, environmental insults, and age-related issues, are linked to the induction of oxidative stress, which could activate expression of ocular HCMV genes, resulting in the production of inflammatory/angiogenic factors and thereby facilitating development of an AMD-like pathology.

Cells and Virus
MCMV strain K181 was originally provided by Dr. Edward Morcarski, Emory University, Atlanta, GA. The virus was prepared from the salivary glands of MCMV-infected immunosuppressed BALB/c mice, and virus stocks were titered on monolayers of mouse embryo fibroblast (MEF) cells, as described previously [69]. A fresh aliquot of virus stocks was thawed and diluted to the appropriate concentration for each experiment.

Mice
We purchased breeding pairs of BALB/c mice from Jackson Laboratory (Bar Harbor, ME). All mice were given unrestricted access to food and water and were maintained on a 12 h light cycle alternating with a 12 h dark cycle. The breeding and treatment of animals in this study adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Institutional Animal Care and Use Committee of Augusta University. The rd8 mutation was excluded by genotyping.

Experimental Design
A total amount of 50 pfu of MCMV or culture medium as control were injected into BALB/c mice within 3 days after birth via the intraperitoneal (i.p.) route. At 18 months post infection (p.i.), the mice were anesthetized, and spectral-domain (SD) optical coherence tomography (SD-OCT) was performed using the Bioptigen Spectral-Domain Ophthalmic Imaging System (En-visu R2200; Bioptigen, Morrisville, NC, USA). The OCT imaging protocol included averaged single B scan and volume intensity scans with images centered on the optic nerve head (1.4 mm × 1.4 mm, @0.0, 1000X100X4X1). Total retinal thickness was measured by manual assessment of retinal layers using InVivoVue™ Diver 2.4 software (Bioptigen) following the software introduction, as described previously [8]. The mice were euthanized, and the eyes were collected and prepared for RNA-Seq, immunofluorescence staining, Western blot, and real time RT-PCR (qRT-PCR) as described below.

RNA Extraction and Quality Control
Six eyes from infected mice at eighteen months p.i., and three eyes from age-matched, uninfected controls were used for RNA sequencing. Following removal of the lens, total RNA was extracted from whole eyes using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and all nine RNA samples were then treated with DNase to exclude genomic DNA contamination. RNA concentrations were obtained using a Nanodrop 2000c spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA), while RNA integrity was assessed using an Agilent 2200 Tape station instrument (Agilent Technologies, Santa Clara, CA, USA. RNA Integrity Number (RIN) scores for the nine samples were 7. 6, 7.7, 7.7, 7.8, 8.0, 8.2, 8.2, 8.8, and 8.9, respectively. One microgram of total RNA from each sample was used to prepare Ribo-Zero RNA-Seq libraries.

Total RNA-Seq with Ribo-Zero Depletion
RNA-Seq libraries were prepared using the Illumina TruSeq Stranded Total RNA kit (Illumina, Inc., San Diego, CA, USA) according to the manufacturer's protocol. Briefly, ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads according to the Illumina Reference Guide (Illumina, San Diego, CA, USA). Following purification, the RNA was fragmented into small pieces using divalent cations at an elevated temperature. First strand cDNA synthesis was performed at 25 • C for 10 min, 42 • C for 15 min, and 70 • C for 15 min, using random hexamers and ProtoScript II Reverse Transcriptase (NEW ENGLAND BioLabs Inc.). For second strand cDNA synthesis, RNA templates were removed, and a second replacement strand was generated through the incorporation of dUTP (in place of dTTP, to maintain strand identity) and double-strand cDNA was generated. Blunt-ended cDNA was isolated from the second strand reaction mix using beads. The three ends of the cDNA were then adenylated, and the cDNA was ligated to indexing adaptors. PCR (15 cycles of 98 • C for 10 s, 60 • C for 30 s, and 72 • C for 30 s) was used to selectively enrich for DNA fragments with adapter molecules on both ends, and to amplify the amount of DNA in the library. Libraries were quantified and qualified using the D1000 Screen Tape on an Agilent 2200 Tape Station instrument and were normalized, pooled, and subjected to cluster and pair read sequencing for 150 cycles on a HiSeqX10 instrument (Illumina, Inc. San Diego, CA, USA), according to the manufacturer's instructions.

Data Analysis Methods
Coding RNA data were analyzed by Rosalind (https://rosalind.onramp.bio/) (accessed on 21 September 2020), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. (San Diego, CA, USA) [70]. Reads were trimmed using cutadapt [71]. Quality scores were assessed using FastQC [72], and reads were aligned to the Mus musculus genome build mm10 using STAR [73]. Individual sample reads were quantified using HTseq [74] and normalized via Relative Log Expression (RLE) using the DESeq2 R library [75]. Read distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC [76]. DEseq2 was also used to calculate fold changes (FDs) and p-values. The significant gene set was selected with a q-value (p-value that has been adjusted for False Discovery Rate, FDR) of <0.05 threshold.
Using QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) (Ingenuity ® Systems, www.ingenuity.com) (accessed on 23 March 2021), we illustrated clustering of genes for the final heatmap of differentially expressed genes and performed functional analyses to identify relevant gene pathways and networks.

qRT-PCR
RNA-seq transcriptome data were validated by qRT-PCR analysis. All primer sequences used for qRT-PCR are shown in Supplementary Table S3. Genes were amplified in 20 µL reaction consisting of 10 µL 2×SYBR Mix (Bio-Rad), 0.2 µL of 20 pmol/µL primer mixture, and 1 µL cDNA, using CFX96TM Real Time PCR System (Bio-Rad). PCR conditions were as follows: 3 min at 94 • C, followed by 40 cycles of 94 • C for 10 s, 60 • C for 20 s, and 72 • C for 30 s. All CT values were analyzed and normalized to β-actin using the method of 2-∆∆CT.

Immunofluorescence Staining
Eyes (four eyes in each group) were embedded in OCT compound, frozen, and sectioned in a cryostat. Sections were then fixed with 4% paraformaldehyde for 15 min and stained by TUNEL assay (in Situ Cell Death Detection Kit, Fluorescein; Roche Diagnostics, Indianapolis, IN, USA) and/or for RPE-65, GFAP, Iba-1 as described previously [77,78].

Western Blot
Lenses were removed from harvested eyes and the remaining eye tissues homogenized in a lysis buffer containing protease inhibitors (Complete™ Lysis-M, Roche, Germany). Proteins were extracted as previously described [79,80], and equal amounts of protein were separated by 10% or 12% SDS-PAGE, followed by electroblotting onto polyvinylidene difluoride membranes (Amersham Biosciences, Amersham, UK). Following blocking with 5% nonfat dry milk for 1 h at room temperature, membranes were incubated overnight at 4 • C with primary antibody. Binding of HRP-conjugated secondary antibody was performed for 1 h at room temperature, and bands were visualized using chemiluminescence (ECL; GE Healthcare, Chicago, IL, USA).